One of the most fundamental aspects of the pharmacology of opioid receptors, as well as other members of the G protein coupled receptor class, is the understanding of "intrinsic activity", the mechanism whereby different agonists produce different maximum responses. Elucidating this process requires a cell system that both expresses a pure receptor type and permits quantitative evaluation of its responses to drugs. In previous years including the current one, we have employed Xenopus oocytes to express the mu opioid receptor, wild-type and mutants, by injecting the appropriate cDNA into the oocyte nucleus. We co-inject the cDNA encoding an inward rectifier potassium channel to which the mu receptor couples to act as a reporter of activation by gating the flow of potassium ions in response to mu opioid agonists. We have employed this system to show that certain mutant varieties of the receptor, namely H297N and H297Q, transduce alkaloids but not peptides to give intrinsic activities that are enhanced in comparison to the wild-type receptor. This enhancement is most conspicuous with antagonists, which behave as partial agonists at the mutant receptors. One theory to account for this change in intrinsic activity predicted that recovery from antagonists would be faster at the mutant receptors, and we confirmed this prediction during the past two years. Over the past year we have attempted to refine and define our system. In vitro transcription seemed to enhance expression. We have also explored the possibilities for inserting 5' untranslated leader sequences to further enhance translation to maximize our chances of success in "macro patch" experiments for very rapid application of drugs using microscopic patches of oocyte membrane. Binding assays on the oocyte membranes were attempted to count receptors. In addition, we have begun an effort to view the receptors directly using fluorescent probes visualized at the laser scanning confocal microscope. We are only the second group to see clear signals from a reversible probe directed at the mu opioid receptor, and we plan to extend these preliminary observations through systematic quantitative kinetic measurements.